Research Article Received: 10 February 2015
Revised: 20 May 2015
Accepted article published: 10 June 2015
Published online in Wiley Online Library: 14 July 2015
(wileyonlinelibrary.com) DOI 10.1002/jsfa.7309
Bioactive compounds of juices from two Brazilian grape cultivars Juliana Kelly da Silva,a Cinthia Baú Betim Cazarin,a Luiz Claudio Correa,b Ângela Giovana Batista,a Cibele Priscila Busch Furlan,a Aline Camarão Telles Biasoto,b Giuliano Elias Pereira,c Adriano Costa de Camargod and Mário Roberto Maróstica Juniora* Abstract BACKGROUND: Grape juice consumption may prevent several chronic diseases owing to the presence of phenolic compounds, which have an important role in the reduction of oxidative stress. This study investigated the polyphenol content and antioxidant activities of grape juices from two cultivars: BRS-Cora and Isabella. Total polyphenol content (TPC), anthocyanins, antioxidant capacity (oxygen radical absorbance capacity, ferric reducing antioxidant power and 1,1-diphenyl-2-picrylhydrazyl), and phenolic profile (high-performance liquid chromatography with diode array and fluorescence detection – HPLC-DAD-FLD) were determined. RESULTS: BRS-Cora grape juice showed higher concentrations of total polyphenols and anthocyanins, as well as higher antioxidant potential, than those of Isabella grape juice. A significant positive correlation was found in TPC or anthocyanin contents when correlated with the remaining antioxidant assays. In addition, HPLC-DAD-FLD showed a higher total phenolic content in BRS-Cora grape juice compared to Isabella. CONCLUSION: The present results show BRS-Cora as a promising cultivar for grape juice production with an improved functional potential. © 2015 Society of Chemical Industry Keywords: Isabella grape; BRS-Cora grape; grape juice; polyphenols; antioxidant potential; bioactive compounds
INTRODUCTION Grapes, grape juice and wine have shown both in vitro and in vivo potential to prevent several chronic diseases. The benefits stem from their high polyphenol content, which are secondary metabolites produced by plants as a defense mechanism against environmental stress.1 – 4 The protective effects against health problems such as cancer and cardiovascular diseases may be attributed to their antioxidant activity and antiproliferative properties.5 Furthermore, interest in polyphenols has increased owing to their potential activity as antitoxic and cytoprotective molecules with a modulatory action in inflammatory processes and neurodegenerative diseases.1 – 3,6,7 The search for natural sources of bioactive compounds is a strong trend nowadays. At the same time, an increase in the consumption of grapes and their products was noted, focusing not only on nutrition but also on their beneficial health effects. Grapes are sources of polyphenols belonging to different subclasses such as monomeric flavan-3-ols, proanthocyanidins, flavonoids, anthocyanins, phenolic acids and the stilbene resveratrol.8 – 11
1990
Several factors such as geographic origin, cultivar and environmental conditions may influence polyphenol concentration.9,12 – 15 Different agronomic strategies have been employed to develop new crops, focusing on sensory quality, field production yield and J Sci Food Agric 2016; 96: 1990–1996
resistance to pests and diseases,16,17 which, in turn, may influence the biosynthesis of phenolic compounds.12 Red wine is the most famous grape product, which has been correlated with the ‘French paradox’, in which low coronary disease death rates have been observed in France, despite the high amount of dietary cholesterol and saturated fatty acids. However, several studies have also reported improvements on health aspects with grape juice intake. Supplementation with Concord grape juice (8 mL kg−1 body weight d−1 ) for 14 days positively altered the endothelial function in adults with coronary artery
∗
Correspondence to: Mário Roberto Maróstica Junior, Departamento de Alimentos e Nutrição, Rua Monteiro Lobato 80, Cidade Universitária, Campinas-SP, Brazil. E-mail: [email protected]
a Department of Food and Nutrition, University of Campinas, Campinas, São Paulo 13083-862, Brazil b Brazilian Agricultural Research Corporation, Embrapa Tropical Semi-arid, Petrolina, Pernambuco 56302-970, Brazil c Brazilian Agricultural Research Corporation, Embrapa Grape Wine/Tropical Semi-arid, Petrolina, Pernambuco 56302-970, Brazil
and
d Department of Agri-Food Industry, Food & Nutrition, ‘Luiz de Queiroz’ College of Agriculture, University of São Paulo, CEP 13418-900, Piracicaba, SP, Brazil
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© 2015 Society of Chemical Industry
Bioactive compounds of juices from two Brazilian grape cultivars disease.18 Castilla et al.19 found greater antioxidant capacity and high-density lipoprotein cholesterol, as well as a reduction in low-density lipoprotein (LDL) cholesterol in plasma of healthy adults and adults in hemodialysis subsequent to the daily ingestion of concentrated grape juice (100 mL for 14 days). In another study, supplementation with 480 mL of grape juice for 8 weeks showed protection against DNA damage in healthy adults of both genders.20 All of these facts indicate that grape juice has the potential to maintain health and collaborate in the control of disease. Grape juice is a representative product of grape processing and there is a constant need for improvement of its sensory quality and nutritional properties. Brazilian grape juice is mainly produced using Isabella cultivar, owing to its large production and the foxy aroma/flavor characteristic of Vitis labrusca cultivars. However, the color of Isabella grape juice generally needs to be improved, as the color is the first sensory attribute evaluated by consumers. The grape BRS-Cora, developed by the Brazilian Agricultural Research Agency (EMBRAPA) via the cross between ‘Muscat Belly A’ and ‘H. 65.9.14’,21 is frequently used to produce grape juice in Brazil, especially to produce blends with Isabella cultivar to improve the final color of the juice. Furthermore, the polyphenol content and antioxidant properties of BRS-Cora are equally important. A quick search of the literature demonstrates that only a few reports have addressed the phenolic profile and antioxidant potential of Isabella cultivar grape juice, and most of them are of local relevance, which also applies to BRS-Cora grape juice. Brazil is very important as a grape juice producer, which makes the present work of international relevance for both researchers and industry. Thus the aim of the present work was to evaluate the phenolic profile and antioxidant properties of grape juices produced with Isabella and BRS-Cora cultivars.
MATERIAL AND METHODS Sample and juice preparation Grapes (cv. Isabella and BRS-Cora) were harvested and provided by Embrapa Tropical Semi-arid, Petrolina, Pernambuco State, Brazil (latitude 9∘ 8′ 8.9′′ S, longitude 40∘ 18′ 33.6′′ W, altitude 373 m). Ripe fruits, which showed mean values of 0.7 g 100 mL−1 , 3.8 and 23 ∘ Brix for total acidity, pH and sugar level, respectively, were harvested and stored at 10 ∘ C for 12 h after processing. First, the grapes were sanitized with hypochlorite solution (100 mg L−1 ) for 15 min and rinsed with water. Fruits were then processed by the steam extraction method for 60 min at 75–85 ∘ C. Sterilized glass bottles were used for juice storage (16 ∘ C) until analysis, within 30 days of juice bottling.
J Sci Food Agric 2016; 96: 1990–1996
Total anthocyanins Total anthocyanins were measured according to Wrolstad,23 with some modifications, as described by Abe et al.24 Appropriate dilutions from the samples were carried out using 0.025 mol L−1 potassium chloride buffer (pH 1.0). The absorbance was read at 510 and 700 nm using a microplate reader (Synergy HT). A second dilution was made for each sample using 0.4 mol L−1 sodium acetate buffer (pH 4.5) and the absorbance was read at the same wavelengths. The calculations were done as shown in the equation below: ) ] [( ) ] [( A510 nm − A700 nm pH = 1.0 − A510 nm − A700 nm pH = 4.5 (1) The results were expressed as mg cyanidin 3-glucoside 100 mL−1 . The final results were calculated as follows: A=
) ( C mg 100 mL−1 = A.MW.DF.100 ÷ 𝜉.1
(2)
where 𝜉 = molar absorptivity (26900 mol L−1 ), 1 = path length (cm), MW = molecular weight of cyanidin 3-glucoside and DF = dilution factor. Analysis by high-performance liquid chromatography (HPLC) Phenolic compounds were identified and quantified by HPLC technique using a Waters Alliance e2695 HPLC system equipped with a photodiode array detector (DAD) and fluorescence detectors (FLD). Separations were performed on a C18 column (150 × 4.6 mm, 3 μm Gemini NX, Phenomenex, Torrance, CA, USA); preceding the analytical column a C18 guard column was used (4.0 × 3.0 mm, Gemini NX) at 40 ∘ C. The mobile phase consisted of a 25 mmol L−1 solution of potassium dihydrogen phosphate in ultrapure water (PURELAB Option Q, Elga Systems, Woodridge, IL, USA) with pH adjusted to 2.05 using phosphoric acid (Fluka, Switzerland) as solvent A, methanol (HPLC grade, JT Baker, Phillipsburg, NJ, USA), as solvent B and acetonitrile (HPLC grade, JT Baker) as solvent C. The elution gradient used was as follows: 0 min 100% A; 18 min 87.5% A, 2.5% B, 10.0% C; 30 min 83.5% A, 3.2% B, 13.3% C; 36 min 75.0% A, 5.0% B, 20.0% C; 48.5 min 65.0% A, 8.3% B, 26.7% C; 50 min 65.0% A, 8.3% B, 26.7% C and 65 min 100% A. The flow rate and injection volume were 0.6 mL min−1 and 10 μL, respectively. The juices were previously filtered through a 0.45 μm membrane before injection into the HPLC system. Phenolic compounds were identified by comparing their relative retention times (RT) and UV spectral data with those of identical standards at 520 nm (pelargonidin 3-O-glucoside, cyanidin 3-O-glucoside, delphinidin 3-O-glucoside, peonidin 3-O-glucoside, malvidin 3-O-glucoside), 220 nm (gallic acid, procyanidin B1, (−)-epicatechin gallate and (−)-epigalocatechin gallate), 320 nm (chlorogenic acid, caffeic acid, p-coumaric acid and ferrulic acid) and 360 nm (isorhamnetin-3-O-glucoside and rutin). The fluorescence detector was used for the detection of the vanillic acid, procyanidin B2 and A2, (−)-epicatechin, (+)-catechin and syringic acid at 280 nm of the excitation and 360 nm of the emission. The standards caffeic, vanillic, ferrulic and gallic acids were purchased from Chem Service (West Chester, PA, USA). Chlorogenic, syringic and p-coumaric acids were purchased from Sigma (UK). Pelargonidin 3-O-glucoside, cyanidin 3-O-glucoside, delphinidin 3-O-glucoside, peonidin 3-O-glucoside, malvidin 3-O-glucoside, procyanidin B1, procyanidin B2, procyanidin A2, epicatechin, catechin, epicatechin gallate, epigalocatechin gallate, rutin and isorhamnetin-3-O-glucoside were obtained from Extrasynthese (Genay, France). Calibration curves were prepared
© 2015 Society of Chemical Industry
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1991
Total polyphenol content (TPC) TPC was determined using the Folin–Ciocalteu method,22 with slight modifications. The extracts, distilled water and Folin–Ciocalteau’s reagent were added to vials and mixed thoroughly. After 3 min, 2 mol L−1 sodium carbonate solution were added and the mixture was kept in the dark at room temperature for 2 h. The absorbance was read at 725 nm using a spectrophotometer (Synergy HT, Biotek, Winooski, VT, USA). Gallic acid was used to prepare a standard curve and the results were expressed as gallic acid equivalents (mg GAE 100 mL−1 of juice).
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www.soci.org for these 21 standard phenolic compounds using the same chromatographic conditions. Table 1 shows the concentration range of each standard used for the calibration curves, standards purity (SP), equations of the calibration curves, coefficients of determination (R2 ), detection limits (LD), quantification limits (LQ) and assay repeatability through coefficient of variation (CV) . Data collection and analysis were carried out using EmporeTM 2 software (Milford, CT, USA). Antioxidant activity DPPH radical scavenging activity The antioxidant activity was determined using the DPPH (1,1-diphenyl-2-picrylhydrazil) assay according to Brand-Williams et al.,25 with some modifications. Briefly, 61 μmol L−1 DPPH ethanol solution were added to the extracts, followed by shaking and incubation for 30 min in the dark. The absorbance was read at 515 nm using a Synergy HT spectrometer. The results were expressed as μmol Trolox equivalents (TE) 100 mL−1 of juice. Hydrophilic oxygen radical absorbance capacity (ORAC) assay The ORAC assay was determined according to Dávalos et al.26 The extracts were prepared in phosphate buffer (PB) (75 mmol L−1 , pH 7.4). The peroxyl radical was generated using 2,2′ -azobis (2-amidinopropane) dihydrochloride solution (AAPH) and fluorescein was used as the substrate. The reaction mixture consisted of extracts, 0.0014 mmol L−1 fluorescein, and 0.4 mol L−1 AAPH. The reaction was carried out in a plate reader (Synergy HT) with filters of emission at 520 nm and excitation at 485 nm. The incubation was conducted at 37 ∘ C. The fluorescence was recorded every minute for 80 min. The Trolox standard was used to prepare a standard curve and the results were expressed as μmol TE 100 mL−1 juice. Ferric reducing antioxidant power (FRAP) assay The FRAP assay was carried out according to Rufino et al.,27 with slight modifications. In the dark, FRAP reagent was prepared with 300 mmol L−1 acetate buffer (pH 3.6), 10 mmol L−1 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) in a 40 mmol L−1 HCl solution and 20 mmol L−1 FeCl3 . Samples or standard solutions, ultrapure water and FRAP reagent were mixed and allowed to react in a water bath for 30 min at 37 ∘ C. After cooling to room temperature, the absorbance of the samples and standards was read at 595 nm. Results were expressed in μmol TE 100 mL−1 juice. Statistical analysis All analyses were carried out in triplicate and data were expressed as the mean value ± standard error (SEM). The statistical analyses were carried out using GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA). The significance of the data was determined using Student’s t-test at P < 0.05.
RESULTS AND DISCUSSION
1992
Total phenolic and anthocyanin content Fruits and plant-derived beverages such as fruit juices are dietary sources of polyphenols. Polyphenols have received increasing interest from consumers and the food industry because of the direct association between their consumption and the prevention of several diseases. Plant polyphenols have antioxidant properties and therefore an important role against radical oxygen species
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JK da Silva et al.
(ROS).28,29 Enhancements in the cognitive function of adults after the ingestion of 6–9 mL grape juice kg−1 body weight for 12 weeks have also been reported.30 Other authors have found positive effects in the lipid profile,19 antioxidant status31 and antimutagenic activity20 after grape juice consumption, which supports the potential health benefits of this product. In the present study, BRS-Cora grape juice showed higher bioactive substance concentrations than that found in grape juice prepared with the Isabella cultivar. BRS-Cora grape juice showed three times higher TPC (221.6 ± 1.83 mg GAE 100 mL−1 ) than Isabella (74.86 ± 4.25 mg GAE 100 mL−1 ) (Fig. 1a). Furthermore, BRS-Cora grape juice also had an anthocyanin content that was five times higher (73.88 ± 1.30 mg cyanidin-3-glucoside 100 mL−1 ) than Isabella grape juice (15.40 ± 0.20 mg cyanidin-3-glucoside 100 mL−1 ) (Fig. 1b). The results presented here are in good agreement with those reported for Campbell Early grape juice, for which TPC was found at a concentration of 308.2 ± 11.0 mg GAE 100 mL−1 and content of 496.6 ± 15.0 mg expressed as malvidin-3-glucoside 100 mL−1 for total anthocyanins 32 . Minor differences are expected due to different cultivars, climate conditions, soil quality and losses during the juice-making process.13 – 15,33,34 Compared to other fruit juices, the analyzed grape juices are rich sources of polyphenol compounds. Pala and Toklucu35 observed a lower anthocyanin content in pomegranate juice (42.6 ± 1.37 mg cyanidin 3-glucoside 100 mL−1 ). HPLC analysis Phenolic compounds are widely distributed in plants, and grapes are well recognized as good sources of polyphenols from several classes. In the present study, 21 phenolic compounds were positively identified and quantified by HPLC (Table 2). Anthocyanins represent a significant portion of the total polyphenols in grapes.32 These compounds are among the most important groups of water-soluble pigments, concentrated mainly in the fruit skin, being responsible for the purplish color of the grape and the antioxidant properties.36 Isabella grape juice showed a higher content of four monoglycosylated anthocyanins (Table 2), but delphinidin 3-O-glucoside was eight times more concentrated in BRS-Cora. Some works have shown prevalence of anthocyanins 3,5-diglucoside in non-vinifera grape cultivars and their hybrids.37 – 39 Diglucosidic anthocyanins were not investigated in the present study; however, as can be noted in Table 2, the phenolic total content (anthocyanins + phenolic acids + flavonoids/proanthocyanidins) was higher in BRS-Cora grape juice, supporting the TPC data. These compounds may be able to avoid certain diseases, thus providing a better life quality.40 The major phenolic compounds of BRS-Cora grape juice were delphinidin 3-O-glucoside, proanthocyanidin B1, ferulic acid and caffeic acid. Proanthocyanidin B1, malvidin 3-O-glucoside, syringic acid and catechin were the major ones in Isabella grape juice. In general, the concentration of individual phenolic compounds was different (P ≤ 0.05) between the samples. BRS-Cora grape juice rendered a higher concentration of most of the phenolics quantified, which is in agreement with the trend observed in TPC and anthocyanin analyses. Among all the phenolic compounds identified, only isorhamnetin and malvidin 3-O-glucoside were not found in BRS-Cora grape juices, and p-coumaric acid and epicatechin gallate were not found in Isabella grape juices. trans-Resveratrol, kaempferol-3-O-glucoside, myricetin and quercetin were not identified in either juice.
© 2015 Society of Chemical Industry
J Sci Food Agric 2016; 96: 1990–1996
Bioactive compounds of juices from two Brazilian grape cultivars
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Table 1. Concentration range (CR, mg L−1 ) of the standards used to prepare the calibration curves, standards purity (SP, %), equations of the calibration curves, coefficients of determination (R2 ), detection limits (LD, mg L−1 ), quantification limits (LQ, mg L−1 ) and assay repeatability through coefficient of variation (CV, %)
Phenolic compound
CR (mg L−1 )
SP (%)
0.625–20 0.625–20 0.625–20 0.625–20 0.625–20 1.25–20 1.25–20 5.00–80 1.25–20 2.5–40 2.5–40 2.5–40 2.5–40 0.625–10 0.625–10 1.25–20 2.50–40 2.50–40 2.50–40 2.50–40 1.25–20
≥98 ≥98 ≥95 ≥95 ≥95 ≥98 ≥98 ≥99 ≥80 ≥98 ≥99 ≥99 ≥97 ≥95 ≥98 ≥98 ≥98 ≥90 ≥95 ≥97 ≥98
1. Pelargonidin 3-O-glucoside 2. Cyanidin 3-O-glucoside 3. Delphinidin 3-O-glucoside 4. Peonidin 3-O-glucoside 5. Malvidin 3-O-glucoside 6. Epicatechin gallate 7. Epigalocatechin gallate 8. Gallic acid 9. Procyanidin B1 10. p-Coumaric acid 11. Caffeic acid 12. Chlorogenic acid 13. Ferrulic acid 14. Isorhamnetin-3-O-glucoside 15. Rutin 16. Procyanidin A2 17. Catechin 18. Procyanidin B2 19. Syringic acid 20. Vanillic acid 21. Epicatechin
Equation of calibration curves Y = 4.26e + 004 X + 2.71e + 003 Y = 6.42e + 004 X + 4.84e + 003 Y = 5.56e + 004 X + 6.28e + 003 Y = 6.35e + 004 X + 4.12e + 003 Y = 3.47e + 004 X + 1.46e + 004 Y = 8.93e + 004 X + 8.55e + 003 Y = 9.64e + 004 X + 3.22e + 003 Y = 1.34e + 005 X + 5.31e + 004 Y = 1.17e + 005 X + 8.54e + 003 Y = 1.09e + 005 X + 1.79e + 004 Y = 9.99e + 004 X + 1.94e + 004 Y = 5.42e + 004 X + 8.96e + 003 Y = 9.30e + 004 X + 1.80e + 004 Y = 3.06e + 004 X + 2.48e + 003 Y = 2.77e + 004 X + 1.84e + 003 Y = 1.66e + 006 X + 5.87e + 005 Y = 4.01e + 006 X - 4.07e + 005 Y = 1.87e + 006 X + 4.44e + 004 Y = 1.91e + 006 X - 7.75e + 004 Y = 1.23e + 007 X + 3.99e + 006 Y = 1.61e + 007 X + 2.94e + 006
R2 0.9996 0.9997 0.9995 0.9995 0.9997 0.9997 0.9999 0.9996 0.9996 0.9997 0.9996 0.9996 0.9996 0.9995 0.9994 0.9972 0.9994 0.9977 0.9998 0.9990 0.9991
LD (mg L−1 ) 0.02 0.04 0.09 0.04 0.27 0.13 0.09 0.12 0.09 0.08 0.08 0.09 0.09 0.03 0.06 0.23 0.06 0.27 0.28 0.30 0.26
LQ (mg L−1 ) 0.05 0.12 0.30 0.13 0.89 0.42 0.31 0.41 0.31 0.27 0.26 0.30 0.30 0.09 0.20 0.76 0.19 0.89 0.93 1.00 0.86
VC (%) 1.21 2.88 3.39 0.62 1.26 1.97 2.32 1.10 1.45 2.79 2.79 1.43 2.19 1.05 2.93 2.83 1.79 1.88 2.30 1.95 2.95
DAD: 220 nm (compounds 6–9); 320 nm (compounds 10–13); 360 nm (compounds 14–15); 520 nm (compounds 1–5) and fluorescent at 280 nm of the excitation and 360 nm of the emission (compounds 16–21).
(a)
(b)
Figure 1. Total polyphenols (a) and anthocyanin contents (b). GAE, gallic acid equivalent. Data are expressed as means ± SEM. Coefficient of variation (CV) for total polyphenols and total anthocyanins were 3.2% and 1.6%, respectively. Asterisks Indicate a significant difference between BRS-Cora and Isabella grape juice, as analyzed by Student’s t-test (***P ≤ 0.001).
J Sci Food Agric 2016; 96: 1990–1996
is the major compound observed in different polyphenol profiles of some grape varieties (Fernão Pires, Castelão, Vital, Vinhão, Espadeiro, Azal Tinto). The present study is in accordance with the literature in relation to the variation in polyphenol composition among different grape cultivars. BRS-Cora was developed by Embrapa, Brazil, with the objective of creating new varieties for grape juice as an alternative to emerging Brazilian regions for grape production, with high productivity, sugar content, color intensity and good sensory quality of grape juice, as well as the Vitis labrusca cultivars Isabella and Concord.44 Therefore, it was expected that BRS Cora grape juice had a higher anthocyanins
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1993
Supporting the findings of the present study, Sensoy41 also found caffeic acid and syringic acids as the major compounds in varieties of red grapes. However, grape juices prepared with Sauvignon Blanc cultivar showed higher catechin and lower caffeic acid concentrations than those found in the present study, within the range of 4.5–7.7 and 0.41–3.7 mg L−1 , respectively.42 Such differences may be correlated with the different grape cultivars compared as the present study, which used red grapes, and these authors used Sauvignon Blanc, which is a white grape. However, Ghafoor et al.32 did not find caffeic acid in grape juice prepared with Campbell Early grape. According to Sun et al.,43 ellagic acid
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JK da Silva et al.
Table 2. Phenolic compounds (mg L−1 ) in BRS-Cora and Isabella grape juices as evaluated by HPLC-DAD-FLD BRS-CORA Anthocyanins Pelargonidin 3-O-glucoside Cyanidin 3-O-glucoside Delphinidin 3-O-glucoside Peonidin 3- O-glucoside Malvidin 3- O-glucoside Σ Anthocyanins Phenolic acids Gallic acid Caffeic acid Syringic acid Vanillic acid Ferulic acid Chlorogenic acid p-coumaric acid Σ Phenolic acids Flavonoids/proanthocyanidins Isorhamnetin 3-O-glucoside Catechin Epicatechin Epicatechin gallate Epigallocatechin gallate Proanthocyanidin A2 Proanthocyanidin B1 Proanthocyanidin B2 Rutin Σ Flavonoids/proanthocyanidins 𝚺 Phenolic compounds (anthocyanins + phenolic acids + flavonoids/ proanthocyanidins)
Isabella
0.33 ± 0.03b 2.77 ± 0.03b 23.3 ± 0.10a 0.55 ± 0.00b ND 26.98 ± 0.09b
3.28 ± 0.03a 3.27 ± 0.03a 2.80 ± 0.50b 6.39 ± 0.05a 23.2 ± 0.18 38.85 ± 0.15a
3.52 ± 0.03a 7.42 ± 0.29a 6.02 ± 0.13b 0.58 ± 0.03a 19.20 ± 1.13a 1.85 ± 0.45 1.32 ± 0.03 39.90 ± 0.51a
1.75 ± 0.00b 1.50 ± 0.00b 11.38 ± 0.40a 0.25 ± 0.00b 0.62 ± 0.03b 1.85 ± 0.00 ND 17.35 ± 0.16b
ND 2.57 ± 0.03b 1.83 ± 0.03a 1.95 ± 0.09 4.65 ± 0.07a 1.15 ± 0.05b 20.78 ± 0.69b 3.33 ± 0.03a 5.42 ± 0.03a 41.68 ± 0.14
0.55 ± 0.00 2.68 ± 0.03a 0.20 ± 0.00b ND 0.48 ± 0.02b 1.35 ± 0.03a 32.57 ± 0.20a 1.92 ± 0.39b 1.63 ± 0.03b 41.38 ± 0.08
108.86 ± 0.37a
97.58 ± 0.17b
Table 3. Pearson’s correlation coefficients (r) between bioactive compounds and antioxidant capacity of BRS-Cora and Isabella grape juices Polyphenols Parameter ORAC r P r2
FRAP
Anthocyanins
DPPH
ORAC
FRAP
DPPH
0.9872 0.9966 0.9989 0.9916 0.9963 0.9992 0.0002
Revised: 20 May 2015
Accepted article published: 10 June 2015
Published online in Wiley Online Library: 14 July 2015
(wileyonlinelibrary.com) DOI 10.1002/jsfa.7309
Bioactive compounds of juices from two Brazilian grape cultivars Juliana Kelly da Silva,a Cinthia Baú Betim Cazarin,a Luiz Claudio Correa,b Ângela Giovana Batista,a Cibele Priscila Busch Furlan,a Aline Camarão Telles Biasoto,b Giuliano Elias Pereira,c Adriano Costa de Camargod and Mário Roberto Maróstica Juniora* Abstract BACKGROUND: Grape juice consumption may prevent several chronic diseases owing to the presence of phenolic compounds, which have an important role in the reduction of oxidative stress. This study investigated the polyphenol content and antioxidant activities of grape juices from two cultivars: BRS-Cora and Isabella. Total polyphenol content (TPC), anthocyanins, antioxidant capacity (oxygen radical absorbance capacity, ferric reducing antioxidant power and 1,1-diphenyl-2-picrylhydrazyl), and phenolic profile (high-performance liquid chromatography with diode array and fluorescence detection – HPLC-DAD-FLD) were determined. RESULTS: BRS-Cora grape juice showed higher concentrations of total polyphenols and anthocyanins, as well as higher antioxidant potential, than those of Isabella grape juice. A significant positive correlation was found in TPC or anthocyanin contents when correlated with the remaining antioxidant assays. In addition, HPLC-DAD-FLD showed a higher total phenolic content in BRS-Cora grape juice compared to Isabella. CONCLUSION: The present results show BRS-Cora as a promising cultivar for grape juice production with an improved functional potential. © 2015 Society of Chemical Industry Keywords: Isabella grape; BRS-Cora grape; grape juice; polyphenols; antioxidant potential; bioactive compounds
INTRODUCTION Grapes, grape juice and wine have shown both in vitro and in vivo potential to prevent several chronic diseases. The benefits stem from their high polyphenol content, which are secondary metabolites produced by plants as a defense mechanism against environmental stress.1 – 4 The protective effects against health problems such as cancer and cardiovascular diseases may be attributed to their antioxidant activity and antiproliferative properties.5 Furthermore, interest in polyphenols has increased owing to their potential activity as antitoxic and cytoprotective molecules with a modulatory action in inflammatory processes and neurodegenerative diseases.1 – 3,6,7 The search for natural sources of bioactive compounds is a strong trend nowadays. At the same time, an increase in the consumption of grapes and their products was noted, focusing not only on nutrition but also on their beneficial health effects. Grapes are sources of polyphenols belonging to different subclasses such as monomeric flavan-3-ols, proanthocyanidins, flavonoids, anthocyanins, phenolic acids and the stilbene resveratrol.8 – 11
1990
Several factors such as geographic origin, cultivar and environmental conditions may influence polyphenol concentration.9,12 – 15 Different agronomic strategies have been employed to develop new crops, focusing on sensory quality, field production yield and J Sci Food Agric 2016; 96: 1990–1996
resistance to pests and diseases,16,17 which, in turn, may influence the biosynthesis of phenolic compounds.12 Red wine is the most famous grape product, which has been correlated with the ‘French paradox’, in which low coronary disease death rates have been observed in France, despite the high amount of dietary cholesterol and saturated fatty acids. However, several studies have also reported improvements on health aspects with grape juice intake. Supplementation with Concord grape juice (8 mL kg−1 body weight d−1 ) for 14 days positively altered the endothelial function in adults with coronary artery
∗
Correspondence to: Mário Roberto Maróstica Junior, Departamento de Alimentos e Nutrição, Rua Monteiro Lobato 80, Cidade Universitária, Campinas-SP, Brazil. E-mail: [email protected]
a Department of Food and Nutrition, University of Campinas, Campinas, São Paulo 13083-862, Brazil b Brazilian Agricultural Research Corporation, Embrapa Tropical Semi-arid, Petrolina, Pernambuco 56302-970, Brazil c Brazilian Agricultural Research Corporation, Embrapa Grape Wine/Tropical Semi-arid, Petrolina, Pernambuco 56302-970, Brazil
and
d Department of Agri-Food Industry, Food & Nutrition, ‘Luiz de Queiroz’ College of Agriculture, University of São Paulo, CEP 13418-900, Piracicaba, SP, Brazil
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Bioactive compounds of juices from two Brazilian grape cultivars disease.18 Castilla et al.19 found greater antioxidant capacity and high-density lipoprotein cholesterol, as well as a reduction in low-density lipoprotein (LDL) cholesterol in plasma of healthy adults and adults in hemodialysis subsequent to the daily ingestion of concentrated grape juice (100 mL for 14 days). In another study, supplementation with 480 mL of grape juice for 8 weeks showed protection against DNA damage in healthy adults of both genders.20 All of these facts indicate that grape juice has the potential to maintain health and collaborate in the control of disease. Grape juice is a representative product of grape processing and there is a constant need for improvement of its sensory quality and nutritional properties. Brazilian grape juice is mainly produced using Isabella cultivar, owing to its large production and the foxy aroma/flavor characteristic of Vitis labrusca cultivars. However, the color of Isabella grape juice generally needs to be improved, as the color is the first sensory attribute evaluated by consumers. The grape BRS-Cora, developed by the Brazilian Agricultural Research Agency (EMBRAPA) via the cross between ‘Muscat Belly A’ and ‘H. 65.9.14’,21 is frequently used to produce grape juice in Brazil, especially to produce blends with Isabella cultivar to improve the final color of the juice. Furthermore, the polyphenol content and antioxidant properties of BRS-Cora are equally important. A quick search of the literature demonstrates that only a few reports have addressed the phenolic profile and antioxidant potential of Isabella cultivar grape juice, and most of them are of local relevance, which also applies to BRS-Cora grape juice. Brazil is very important as a grape juice producer, which makes the present work of international relevance for both researchers and industry. Thus the aim of the present work was to evaluate the phenolic profile and antioxidant properties of grape juices produced with Isabella and BRS-Cora cultivars.
MATERIAL AND METHODS Sample and juice preparation Grapes (cv. Isabella and BRS-Cora) were harvested and provided by Embrapa Tropical Semi-arid, Petrolina, Pernambuco State, Brazil (latitude 9∘ 8′ 8.9′′ S, longitude 40∘ 18′ 33.6′′ W, altitude 373 m). Ripe fruits, which showed mean values of 0.7 g 100 mL−1 , 3.8 and 23 ∘ Brix for total acidity, pH and sugar level, respectively, were harvested and stored at 10 ∘ C for 12 h after processing. First, the grapes were sanitized with hypochlorite solution (100 mg L−1 ) for 15 min and rinsed with water. Fruits were then processed by the steam extraction method for 60 min at 75–85 ∘ C. Sterilized glass bottles were used for juice storage (16 ∘ C) until analysis, within 30 days of juice bottling.
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Total anthocyanins Total anthocyanins were measured according to Wrolstad,23 with some modifications, as described by Abe et al.24 Appropriate dilutions from the samples were carried out using 0.025 mol L−1 potassium chloride buffer (pH 1.0). The absorbance was read at 510 and 700 nm using a microplate reader (Synergy HT). A second dilution was made for each sample using 0.4 mol L−1 sodium acetate buffer (pH 4.5) and the absorbance was read at the same wavelengths. The calculations were done as shown in the equation below: ) ] [( ) ] [( A510 nm − A700 nm pH = 1.0 − A510 nm − A700 nm pH = 4.5 (1) The results were expressed as mg cyanidin 3-glucoside 100 mL−1 . The final results were calculated as follows: A=
) ( C mg 100 mL−1 = A.MW.DF.100 ÷ 𝜉.1
(2)
where 𝜉 = molar absorptivity (26900 mol L−1 ), 1 = path length (cm), MW = molecular weight of cyanidin 3-glucoside and DF = dilution factor. Analysis by high-performance liquid chromatography (HPLC) Phenolic compounds were identified and quantified by HPLC technique using a Waters Alliance e2695 HPLC system equipped with a photodiode array detector (DAD) and fluorescence detectors (FLD). Separations were performed on a C18 column (150 × 4.6 mm, 3 μm Gemini NX, Phenomenex, Torrance, CA, USA); preceding the analytical column a C18 guard column was used (4.0 × 3.0 mm, Gemini NX) at 40 ∘ C. The mobile phase consisted of a 25 mmol L−1 solution of potassium dihydrogen phosphate in ultrapure water (PURELAB Option Q, Elga Systems, Woodridge, IL, USA) with pH adjusted to 2.05 using phosphoric acid (Fluka, Switzerland) as solvent A, methanol (HPLC grade, JT Baker, Phillipsburg, NJ, USA), as solvent B and acetonitrile (HPLC grade, JT Baker) as solvent C. The elution gradient used was as follows: 0 min 100% A; 18 min 87.5% A, 2.5% B, 10.0% C; 30 min 83.5% A, 3.2% B, 13.3% C; 36 min 75.0% A, 5.0% B, 20.0% C; 48.5 min 65.0% A, 8.3% B, 26.7% C; 50 min 65.0% A, 8.3% B, 26.7% C and 65 min 100% A. The flow rate and injection volume were 0.6 mL min−1 and 10 μL, respectively. The juices were previously filtered through a 0.45 μm membrane before injection into the HPLC system. Phenolic compounds were identified by comparing their relative retention times (RT) and UV spectral data with those of identical standards at 520 nm (pelargonidin 3-O-glucoside, cyanidin 3-O-glucoside, delphinidin 3-O-glucoside, peonidin 3-O-glucoside, malvidin 3-O-glucoside), 220 nm (gallic acid, procyanidin B1, (−)-epicatechin gallate and (−)-epigalocatechin gallate), 320 nm (chlorogenic acid, caffeic acid, p-coumaric acid and ferrulic acid) and 360 nm (isorhamnetin-3-O-glucoside and rutin). The fluorescence detector was used for the detection of the vanillic acid, procyanidin B2 and A2, (−)-epicatechin, (+)-catechin and syringic acid at 280 nm of the excitation and 360 nm of the emission. The standards caffeic, vanillic, ferrulic and gallic acids were purchased from Chem Service (West Chester, PA, USA). Chlorogenic, syringic and p-coumaric acids were purchased from Sigma (UK). Pelargonidin 3-O-glucoside, cyanidin 3-O-glucoside, delphinidin 3-O-glucoside, peonidin 3-O-glucoside, malvidin 3-O-glucoside, procyanidin B1, procyanidin B2, procyanidin A2, epicatechin, catechin, epicatechin gallate, epigalocatechin gallate, rutin and isorhamnetin-3-O-glucoside were obtained from Extrasynthese (Genay, France). Calibration curves were prepared
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Total polyphenol content (TPC) TPC was determined using the Folin–Ciocalteu method,22 with slight modifications. The extracts, distilled water and Folin–Ciocalteau’s reagent were added to vials and mixed thoroughly. After 3 min, 2 mol L−1 sodium carbonate solution were added and the mixture was kept in the dark at room temperature for 2 h. The absorbance was read at 725 nm using a spectrophotometer (Synergy HT, Biotek, Winooski, VT, USA). Gallic acid was used to prepare a standard curve and the results were expressed as gallic acid equivalents (mg GAE 100 mL−1 of juice).
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www.soci.org for these 21 standard phenolic compounds using the same chromatographic conditions. Table 1 shows the concentration range of each standard used for the calibration curves, standards purity (SP), equations of the calibration curves, coefficients of determination (R2 ), detection limits (LD), quantification limits (LQ) and assay repeatability through coefficient of variation (CV) . Data collection and analysis were carried out using EmporeTM 2 software (Milford, CT, USA). Antioxidant activity DPPH radical scavenging activity The antioxidant activity was determined using the DPPH (1,1-diphenyl-2-picrylhydrazil) assay according to Brand-Williams et al.,25 with some modifications. Briefly, 61 μmol L−1 DPPH ethanol solution were added to the extracts, followed by shaking and incubation for 30 min in the dark. The absorbance was read at 515 nm using a Synergy HT spectrometer. The results were expressed as μmol Trolox equivalents (TE) 100 mL−1 of juice. Hydrophilic oxygen radical absorbance capacity (ORAC) assay The ORAC assay was determined according to Dávalos et al.26 The extracts were prepared in phosphate buffer (PB) (75 mmol L−1 , pH 7.4). The peroxyl radical was generated using 2,2′ -azobis (2-amidinopropane) dihydrochloride solution (AAPH) and fluorescein was used as the substrate. The reaction mixture consisted of extracts, 0.0014 mmol L−1 fluorescein, and 0.4 mol L−1 AAPH. The reaction was carried out in a plate reader (Synergy HT) with filters of emission at 520 nm and excitation at 485 nm. The incubation was conducted at 37 ∘ C. The fluorescence was recorded every minute for 80 min. The Trolox standard was used to prepare a standard curve and the results were expressed as μmol TE 100 mL−1 juice. Ferric reducing antioxidant power (FRAP) assay The FRAP assay was carried out according to Rufino et al.,27 with slight modifications. In the dark, FRAP reagent was prepared with 300 mmol L−1 acetate buffer (pH 3.6), 10 mmol L−1 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) in a 40 mmol L−1 HCl solution and 20 mmol L−1 FeCl3 . Samples or standard solutions, ultrapure water and FRAP reagent were mixed and allowed to react in a water bath for 30 min at 37 ∘ C. After cooling to room temperature, the absorbance of the samples and standards was read at 595 nm. Results were expressed in μmol TE 100 mL−1 juice. Statistical analysis All analyses were carried out in triplicate and data were expressed as the mean value ± standard error (SEM). The statistical analyses were carried out using GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA). The significance of the data was determined using Student’s t-test at P < 0.05.
RESULTS AND DISCUSSION
1992
Total phenolic and anthocyanin content Fruits and plant-derived beverages such as fruit juices are dietary sources of polyphenols. Polyphenols have received increasing interest from consumers and the food industry because of the direct association between their consumption and the prevention of several diseases. Plant polyphenols have antioxidant properties and therefore an important role against radical oxygen species
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(ROS).28,29 Enhancements in the cognitive function of adults after the ingestion of 6–9 mL grape juice kg−1 body weight for 12 weeks have also been reported.30 Other authors have found positive effects in the lipid profile,19 antioxidant status31 and antimutagenic activity20 after grape juice consumption, which supports the potential health benefits of this product. In the present study, BRS-Cora grape juice showed higher bioactive substance concentrations than that found in grape juice prepared with the Isabella cultivar. BRS-Cora grape juice showed three times higher TPC (221.6 ± 1.83 mg GAE 100 mL−1 ) than Isabella (74.86 ± 4.25 mg GAE 100 mL−1 ) (Fig. 1a). Furthermore, BRS-Cora grape juice also had an anthocyanin content that was five times higher (73.88 ± 1.30 mg cyanidin-3-glucoside 100 mL−1 ) than Isabella grape juice (15.40 ± 0.20 mg cyanidin-3-glucoside 100 mL−1 ) (Fig. 1b). The results presented here are in good agreement with those reported for Campbell Early grape juice, for which TPC was found at a concentration of 308.2 ± 11.0 mg GAE 100 mL−1 and content of 496.6 ± 15.0 mg expressed as malvidin-3-glucoside 100 mL−1 for total anthocyanins 32 . Minor differences are expected due to different cultivars, climate conditions, soil quality and losses during the juice-making process.13 – 15,33,34 Compared to other fruit juices, the analyzed grape juices are rich sources of polyphenol compounds. Pala and Toklucu35 observed a lower anthocyanin content in pomegranate juice (42.6 ± 1.37 mg cyanidin 3-glucoside 100 mL−1 ). HPLC analysis Phenolic compounds are widely distributed in plants, and grapes are well recognized as good sources of polyphenols from several classes. In the present study, 21 phenolic compounds were positively identified and quantified by HPLC (Table 2). Anthocyanins represent a significant portion of the total polyphenols in grapes.32 These compounds are among the most important groups of water-soluble pigments, concentrated mainly in the fruit skin, being responsible for the purplish color of the grape and the antioxidant properties.36 Isabella grape juice showed a higher content of four monoglycosylated anthocyanins (Table 2), but delphinidin 3-O-glucoside was eight times more concentrated in BRS-Cora. Some works have shown prevalence of anthocyanins 3,5-diglucoside in non-vinifera grape cultivars and their hybrids.37 – 39 Diglucosidic anthocyanins were not investigated in the present study; however, as can be noted in Table 2, the phenolic total content (anthocyanins + phenolic acids + flavonoids/proanthocyanidins) was higher in BRS-Cora grape juice, supporting the TPC data. These compounds may be able to avoid certain diseases, thus providing a better life quality.40 The major phenolic compounds of BRS-Cora grape juice were delphinidin 3-O-glucoside, proanthocyanidin B1, ferulic acid and caffeic acid. Proanthocyanidin B1, malvidin 3-O-glucoside, syringic acid and catechin were the major ones in Isabella grape juice. In general, the concentration of individual phenolic compounds was different (P ≤ 0.05) between the samples. BRS-Cora grape juice rendered a higher concentration of most of the phenolics quantified, which is in agreement with the trend observed in TPC and anthocyanin analyses. Among all the phenolic compounds identified, only isorhamnetin and malvidin 3-O-glucoside were not found in BRS-Cora grape juices, and p-coumaric acid and epicatechin gallate were not found in Isabella grape juices. trans-Resveratrol, kaempferol-3-O-glucoside, myricetin and quercetin were not identified in either juice.
© 2015 Society of Chemical Industry
J Sci Food Agric 2016; 96: 1990–1996
Bioactive compounds of juices from two Brazilian grape cultivars
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Table 1. Concentration range (CR, mg L−1 ) of the standards used to prepare the calibration curves, standards purity (SP, %), equations of the calibration curves, coefficients of determination (R2 ), detection limits (LD, mg L−1 ), quantification limits (LQ, mg L−1 ) and assay repeatability through coefficient of variation (CV, %)
Phenolic compound
CR (mg L−1 )
SP (%)
0.625–20 0.625–20 0.625–20 0.625–20 0.625–20 1.25–20 1.25–20 5.00–80 1.25–20 2.5–40 2.5–40 2.5–40 2.5–40 0.625–10 0.625–10 1.25–20 2.50–40 2.50–40 2.50–40 2.50–40 1.25–20
≥98 ≥98 ≥95 ≥95 ≥95 ≥98 ≥98 ≥99 ≥80 ≥98 ≥99 ≥99 ≥97 ≥95 ≥98 ≥98 ≥98 ≥90 ≥95 ≥97 ≥98
1. Pelargonidin 3-O-glucoside 2. Cyanidin 3-O-glucoside 3. Delphinidin 3-O-glucoside 4. Peonidin 3-O-glucoside 5. Malvidin 3-O-glucoside 6. Epicatechin gallate 7. Epigalocatechin gallate 8. Gallic acid 9. Procyanidin B1 10. p-Coumaric acid 11. Caffeic acid 12. Chlorogenic acid 13. Ferrulic acid 14. Isorhamnetin-3-O-glucoside 15. Rutin 16. Procyanidin A2 17. Catechin 18. Procyanidin B2 19. Syringic acid 20. Vanillic acid 21. Epicatechin
Equation of calibration curves Y = 4.26e + 004 X + 2.71e + 003 Y = 6.42e + 004 X + 4.84e + 003 Y = 5.56e + 004 X + 6.28e + 003 Y = 6.35e + 004 X + 4.12e + 003 Y = 3.47e + 004 X + 1.46e + 004 Y = 8.93e + 004 X + 8.55e + 003 Y = 9.64e + 004 X + 3.22e + 003 Y = 1.34e + 005 X + 5.31e + 004 Y = 1.17e + 005 X + 8.54e + 003 Y = 1.09e + 005 X + 1.79e + 004 Y = 9.99e + 004 X + 1.94e + 004 Y = 5.42e + 004 X + 8.96e + 003 Y = 9.30e + 004 X + 1.80e + 004 Y = 3.06e + 004 X + 2.48e + 003 Y = 2.77e + 004 X + 1.84e + 003 Y = 1.66e + 006 X + 5.87e + 005 Y = 4.01e + 006 X - 4.07e + 005 Y = 1.87e + 006 X + 4.44e + 004 Y = 1.91e + 006 X - 7.75e + 004 Y = 1.23e + 007 X + 3.99e + 006 Y = 1.61e + 007 X + 2.94e + 006
R2 0.9996 0.9997 0.9995 0.9995 0.9997 0.9997 0.9999 0.9996 0.9996 0.9997 0.9996 0.9996 0.9996 0.9995 0.9994 0.9972 0.9994 0.9977 0.9998 0.9990 0.9991
LD (mg L−1 ) 0.02 0.04 0.09 0.04 0.27 0.13 0.09 0.12 0.09 0.08 0.08 0.09 0.09 0.03 0.06 0.23 0.06 0.27 0.28 0.30 0.26
LQ (mg L−1 ) 0.05 0.12 0.30 0.13 0.89 0.42 0.31 0.41 0.31 0.27 0.26 0.30 0.30 0.09 0.20 0.76 0.19 0.89 0.93 1.00 0.86
VC (%) 1.21 2.88 3.39 0.62 1.26 1.97 2.32 1.10 1.45 2.79 2.79 1.43 2.19 1.05 2.93 2.83 1.79 1.88 2.30 1.95 2.95
DAD: 220 nm (compounds 6–9); 320 nm (compounds 10–13); 360 nm (compounds 14–15); 520 nm (compounds 1–5) and fluorescent at 280 nm of the excitation and 360 nm of the emission (compounds 16–21).
(a)
(b)
Figure 1. Total polyphenols (a) and anthocyanin contents (b). GAE, gallic acid equivalent. Data are expressed as means ± SEM. Coefficient of variation (CV) for total polyphenols and total anthocyanins were 3.2% and 1.6%, respectively. Asterisks Indicate a significant difference between BRS-Cora and Isabella grape juice, as analyzed by Student’s t-test (***P ≤ 0.001).
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is the major compound observed in different polyphenol profiles of some grape varieties (Fernão Pires, Castelão, Vital, Vinhão, Espadeiro, Azal Tinto). The present study is in accordance with the literature in relation to the variation in polyphenol composition among different grape cultivars. BRS-Cora was developed by Embrapa, Brazil, with the objective of creating new varieties for grape juice as an alternative to emerging Brazilian regions for grape production, with high productivity, sugar content, color intensity and good sensory quality of grape juice, as well as the Vitis labrusca cultivars Isabella and Concord.44 Therefore, it was expected that BRS Cora grape juice had a higher anthocyanins
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Supporting the findings of the present study, Sensoy41 also found caffeic acid and syringic acids as the major compounds in varieties of red grapes. However, grape juices prepared with Sauvignon Blanc cultivar showed higher catechin and lower caffeic acid concentrations than those found in the present study, within the range of 4.5–7.7 and 0.41–3.7 mg L−1 , respectively.42 Such differences may be correlated with the different grape cultivars compared as the present study, which used red grapes, and these authors used Sauvignon Blanc, which is a white grape. However, Ghafoor et al.32 did not find caffeic acid in grape juice prepared with Campbell Early grape. According to Sun et al.,43 ellagic acid
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Table 2. Phenolic compounds (mg L−1 ) in BRS-Cora and Isabella grape juices as evaluated by HPLC-DAD-FLD BRS-CORA Anthocyanins Pelargonidin 3-O-glucoside Cyanidin 3-O-glucoside Delphinidin 3-O-glucoside Peonidin 3- O-glucoside Malvidin 3- O-glucoside Σ Anthocyanins Phenolic acids Gallic acid Caffeic acid Syringic acid Vanillic acid Ferulic acid Chlorogenic acid p-coumaric acid Σ Phenolic acids Flavonoids/proanthocyanidins Isorhamnetin 3-O-glucoside Catechin Epicatechin Epicatechin gallate Epigallocatechin gallate Proanthocyanidin A2 Proanthocyanidin B1 Proanthocyanidin B2 Rutin Σ Flavonoids/proanthocyanidins 𝚺 Phenolic compounds (anthocyanins + phenolic acids + flavonoids/ proanthocyanidins)
Isabella
0.33 ± 0.03b 2.77 ± 0.03b 23.3 ± 0.10a 0.55 ± 0.00b ND 26.98 ± 0.09b
3.28 ± 0.03a 3.27 ± 0.03a 2.80 ± 0.50b 6.39 ± 0.05a 23.2 ± 0.18 38.85 ± 0.15a
3.52 ± 0.03a 7.42 ± 0.29a 6.02 ± 0.13b 0.58 ± 0.03a 19.20 ± 1.13a 1.85 ± 0.45 1.32 ± 0.03 39.90 ± 0.51a
1.75 ± 0.00b 1.50 ± 0.00b 11.38 ± 0.40a 0.25 ± 0.00b 0.62 ± 0.03b 1.85 ± 0.00 ND 17.35 ± 0.16b
ND 2.57 ± 0.03b 1.83 ± 0.03a 1.95 ± 0.09 4.65 ± 0.07a 1.15 ± 0.05b 20.78 ± 0.69b 3.33 ± 0.03a 5.42 ± 0.03a 41.68 ± 0.14
0.55 ± 0.00 2.68 ± 0.03a 0.20 ± 0.00b ND 0.48 ± 0.02b 1.35 ± 0.03a 32.57 ± 0.20a 1.92 ± 0.39b 1.63 ± 0.03b 41.38 ± 0.08
108.86 ± 0.37a
97.58 ± 0.17b
Table 3. Pearson’s correlation coefficients (r) between bioactive compounds and antioxidant capacity of BRS-Cora and Isabella grape juices Polyphenols Parameter ORAC r P r2
FRAP
Anthocyanins
DPPH
ORAC
FRAP
DPPH
0.9872 0.9966 0.9989 0.9916 0.9963 0.9992 0.0002
